Development of a novel visual assay for ultrasensitive detection of Listeria monocytogenes in milk and chicken meat harnessing helix loop-mediated isothermal amplification (HAMP)

Listeria monocytogenes causes foodborne listeriosis, which is an important food safety and public health problem. Monitoring them in the food chain using simple and affordable methods is of paramount importance to avoid untoward consequences. In this study, we have developed and evaluated a novel helix loop-mediated isothermal amplification (HAMP) assay integrating simple three-step centrifugation DNA isolation and pre-added hydroxynaphthol blue (HNB) dye-based result interpretation. Specific amplification of the hlyA gene of L. monocytogenes was ascertained by deploying reference and field strains of L. monocytogenes (n = 16), non-monocytogenes Listeria (n = 3) and other bacteria (n = 15). The analytical sensitivity of HAMP, real-time, and end-point PCR methods was 5.4 fg/& mu;L, 540 fg/& mu;L and 5.4 pg/& mu;L, respectively, displaying 100x more sensitivity than real-time PCR. The detection limit (LoD) of the developed HAMP assay was determined by analysing milk and chicken meat artificially spiked with ten-fold sequential dilutions of L. monocytogenes. The influence of brief enrichment (3 h/6 h) in LoD was also analysed and compared with endpoint and real-time PCR assays. In artificially contaminated milk, the limits of detection of the HAMP assay were 120 CFU/mL, 12 CFU/mL, and 1.2 CFU/mL without any enrichment, after 3 h and 6 h enrichment, respectively. Whereas in artificially contaminated chicken meat, the detection limits without any enrichment, after 3 h and 6 h enrichment were 1500 CFU/g, 150 CFU/g, and 15 CFU/g, respectively. The real-world applicability of the HAMP assay was also assessed by screening field milk and meat samples (n = 200). The developed HAMP assay is more simple, rapid, sensitive, and specific for the detection of L. monocytogenes in food samples, especially in resource-poor establishments. To our knowledge, this is the first study to develop a HAMP assay for the detection of a bacterial pathogen.